1. Field of the Invention
This invention relates to the field of gene therapy and vaccine delivery, and provides a significant advance in the art by facilitating repeat administration of a transgene in a vector, circumventing anti-vector immune responses, which diminish the efficacy of known gene delivery vectors.
2. Background of the Invention
Recently, adenovirus (Ads) vectors have received considerable attention for transgene delivery to mammalian cells generally and for gene therapy in particular due to several advantages over other vector systems, including high transduction efficiency of a variety of cell types comprised of both replicating and non-replicating cells, ease of growth, and relative safety (for review see Hitt et al. 1997). However, data from preclinical and clinical studies have shown that Ads also have several disadvantages, primarily due to the induction of both cellular and humoral immune responses to vector-derived antigens (Yang et al. 1994a, 1995a,b, 1996a,b, Dai et al. 1995, Gilgenkrantz et al. 1995, McCoy et al. 1995, Christ et al. 1997, Morral et al. 1997, van Ginkel et al. 1997, Kafri et al. 1998). Because of these immune responses, administration of first-generation Ad vectors (i.e. deleted of early region 1 (E1) or E1/E3) has generally resulted in only transient transgene expression and poor expression following repeat vector administration (Dong et al. 1996, Kaplan et al. 1996, St. George et al. 1996, Schulick et al. 1997). Reintroduction of the E3 region, which encodes functions involved in aiding virus escape from host immune responses, can prolong transgene expression in some animal models (Lee et al. 1995, Poller et al. 1996, Bruder et al. 1997, Ilan et al. 1997, Schowalter et al. 1997), and is reported to decrease the formation of anti-Ad neutralizing antibodies (Ilan et al. 1997). The use of second-generation Ad vectors, which are further deleted or attenuated in E2 or E4, can lead to decreased inflammatory responses and a longer duration of transgene expression (Engelhardt et al. 1994, Yang et al. 1994b, Goldman et al. 1995, Gao et al. 1996, Dedieu et al. 1997, Wang et al. 1997, Amalfitano et al. 1998), although not in all cases (Fang et al. 1996, Christ et al. 1997, Morral et al. 1997, Lusky et al. 1998). However induced antibody titers were similar to those generated against first generation vectors (Christ et al. 1997), thus compromising the ability to readminister the vector.
The development of systems for the generation of helper-dependent Ad vectors (hdAd) which are deleted for most if not all viral coding sequences (Mitani et al. 1995, Fisher et al. 1996, Haeker et al. 1996, Kochanek et al. 1996, Kumar-Singh and Chamberlain 1996, Lieber et al. 1996, Parks et al. 1996, Hardy et al. 1997, reviewed by Hitt et al. 1998), has allowed production of hdAd which can provide long term, high level transgene expression (Chen et al. 1997, Schiedner et al. 1998, Morsy et al. 1998), and which result in substantially reduced inflammatory and cellular immune responses (Morsy et al. 1998, Schiedner et al. 1998, Morral et al. 1998). However, as expected, deletion of all Ad coding sequences does not overcome the humoral immune response, and neutralizing antibodies are formed (J. L. Bramson, R. J. P. and F. L. G., unpublished results), thus reducing the effectiveness of hdAd vector readministration.
In an attempt to prevent vector-directed immune responses, many groups have explored the use of transient immune blockage at the time of Ad vector administration, or the induction of tolerance to Ad (Vilquin et al. 1995, Jooss et al. 1996, Kass-Eisler et al. 1996, Kolls et al. 1996, Lochmuller et al. 1996, Sawchuk et al. 1996, Smith et al. 1996, Yang et al. 1996c, Zepeda and Wilson 1996, Kaplan and Smith 1997, Kuzmin et al. 1997, Lieber et al. 1997, Scaria et al. 1997, Wolff et al. 1997, Zsengeller et al. 1997). These strategies have been somewhat successful, and allow repeat vector administration; however, complications and potential side-effects may make immune suppression impractical for clinical use. An alternative strategy to allow for repeat vector administration is the sequential use of different Ad serotypes. Neutralizing antibodies formed against one serotpe should have no effect on subsequent delivery of a different serotype, and this approach has allowed repeat administration of first generation Ad vectors (Kass-Eisler et al. 1996, Mastrangeli et al. 1996, Mack et al. 1997, Roy et al. 1998, A. L. Beaudet, unpublished results). Over 40 different serotypes of human Ads have been isolated, suggesting that, in theory, Ad vectors of different serotypes could be administered many times throughout the life of a patient. However, how to achieve this feat does not appear to have been disclosed or suggested for helper dependent vectors.
The Cre/loxP system for producing helper-dependent Ad vectors involves the use of a helper virus that contains a packaging signal flanked by loxP sites (Parks et al. 1996). Upon infection of a 293-derived cell line that stably expresses the bacteriophage P1 Cre recombinase (Chen et al. 1996), the packaging signal is excised from the helper virus DNA, rendering it unpackageable. The helper virus DNA retains the ability to replicate and provides all of the functions required in trans for the replication and packaging of a hdAd. This system facilitates the generation of high titer hdAd preparations with substantially reduced quantities of contaminating helper-virus. A key feature of the helper-dependent system is that the serotype of the hdAd is determined only by the helper virus. Therefore, in contrast to first generation vectors that require the construction of a new vector to switch serotypes, a series of genetically identical hdAds of different serotypes could be generated simply by changing the serotype of the helper.
We have developed a new helper adenovirus (Ad) based on serotype 2, Ad2LC8cCARP, for use in a the Cre/loxP system (Parks et al. 1996, Proc. Natl. Acad. Sci. USA 93:13565-13570) to generate Ad vectors deleted of all protein coding sequences (helper-dependent Ad vectors (hdAd)). A comparison of Ad2LC8cCARP and our helper virus developed previously (based on serotype 5, Ad5LC8cluc) showed that the two helper viruses amplified hdAd with a similar efficiency, and resulted in a similar yield and purity after large scale preparation of vector. In vitro, the resulting hdAd2 had a similar transduction efficiency and expressed levels of transgene (xcex2-gal) identical to those produced by hdAd5. An important feature of the helper-dependent system is that all virion components, except the virion DNA, derive from the helper virus. Consequently vectors produced with help from Ad2LC8cCARP were not neutralized by antibodies against Ad5, and vectors produced with Ad5 helper were resistant to neutralizing antibodies against Ad2. Analysis of transgene expression in vivo after transduction of mouse liver by intravenous injection of the Ad2-based hdAd showed that the vector could efficiently transduce hepatocytes, and produce high levels of a foreign transgene (human secreted alkaline phosphatase), similar to those expressed by the hdAd generated with the Ad5 helper virus. Mice immunized with hdAd2 produced Ad2-neutralizing antibodies, which did not cross-react with hdAd5. To determine if successful repeat Ad vector administration could be achieved by sequential use of alternative Ad serotypes, we injected mice with hdAd2 (hSEAP) followed three months later by a lacZ-expressing hdAd of either the same or different serotype. Administration of a vector of the same serotype resulted in a 30- to 100-fold reduction in transgene expression compared to naive animals. In contrast, no decrease in transgene expression was observed when the second vector was of a different serotype. These results suggest that effective vector readministration can be achieved by the sequential use of hdAds based on alternative serotypes.
Accordingly, this invention responds to a long felt need, this invention provides, in one embodiment, a helper virus based on the Ad2serotype for use in the Cre/loxP system for the generation of Ad vectors deleted of all Ad protein coding sequences. Using this and helper virus based on Ad5, genetically identical hdAd that differ only in the virion protein components, which are derived from the helper virus, were produced. The vectors have identical expression characteristics in vitro, regardless of the serotype, and the sequential use of hdAd of different serotypes allows for successful repeat vector administration in vivo.
Accordingly, it is one object of this invention to provide a helper-dependent adenovirus vector (hdAd) administration system whereby repeat administration of a gene of interest is facilitated by using hdAd wherein all protein present in said hdAd is derived from a helper virus, the serotype of which is switched in the production of a vector to be used in a repeat hdAd administration.
Another object of this invention is to provide a generally applicable strategy, not restricted to adenoviruses, whereby repeat administration of a gene is facilitated, such that high level gene expression occurs on each administration.
Another object of this invention is to provide a system whereby helper adenoviruses of different serotypes are used to generate a series of hdAd vectors against which humoral and cellular immune responses are minimized, while providing for repeat administration of genes of interest.
Other objects and advantages of this invention will become apparent from a review of the complete disclosure and the appended claims.